Construction Of InlK Gene Deletion Strain Of Listeria Monocytogenes And Correlation Analysis Of Disinfectant Bactericidal Efficiency

Object:In this experiment,Listeria monocytogenes Lm90SB2 was used as the test bacteria to construct the inlK gene deletion strain.To study some biological characteristics of inlK gene in the process of causing Listeria monocytogenes,and analyze the bactericidal efficiency of disinfectants on the biofilms of parental strains and deletion strains,in order to provide a certain reference for effective prevention and control of LM pollution.Methods:Using Listeria monocytogenes Lm90SB2 as the test bacteria,according to the inlK gene sequence of LM F2365（accession number:AE017262）published in Gen Bank,the specific primers required for the design test were carried out;The upstream and downstream homology arms of the inlK gene were amplified by PCR,and theΔinlK fusion fragment was obtained by overlapping extension PCR;Construction of p KSV7-ΔinlK recombinant shuttle plasmid;Preparation of Lm90SB2 competent cells;The p KSV7-ΔinlK was electroporated into Lm90SB2 competent cells by electroporation,and then continuously subcultured at 42°C under the double pressure of chloramphenicol resistance to carry out homologous recombination to obtain the inlK gene deletion strain,which was used by PCR method.Paralateral primers for deletion strain detection;The deletion strain was cultured at 30℃in BHI liquid medium without chloramphenicol,so that the p KSV7 plasmid was lost,and a gene deletion strain without chloramphenicol resistance was obtained;The Lm90SB2 parental strain and the Lm90SB2-ΔinlK deletion strain were compared in the biofilm formation ability,growth difference and hemolytic activity at 37°C at 8,12,24,and 48h,and the LD50 and brain and spleen tissue load were measured by infecting healthy mice.Bacterial quantity,to study the relationship between inlK gene and some biological characteristics of LM.Refer to the"Technical Specification for Disinfection"to screen the neutralizing agent of Neogermin and 84disinfectant,and detect the effects of different concentrations of Neogermin（1:15,1:30）and 84 disinfectant（1:50,1:100）respectively 1,5,10,20min bactericidal rate and removal rate of two bacterial suspensions and biofilm.Results:A stably inherited Lm90SB2-ΔinlK gene deletion strain was successfully obtained by homologous recombination technology;Through the comparison of some biological characteristics of the parental strain and the deletion strain,it was found that the deletion of inlK gene resulted in a very significant decrease in the biofilm formation ability of the Lm90SB2 strain（P<0.01）;After measuring the growth curves of the parental strain and the missing strain,it was found that the growth trends of the two strains were the same,and there was no significant change;The hemolytic activity test showed that the hemolytic value of the parent strain Lm90SB2 was 2¹,and the hemolytic value of the Lm90SB2-ΔinlK gene deletion strain was 2²,indicating that the deletion of inlK gene has a certain influence on the hemolytic ability of Lm90SB2;The mouse infection test found that the median lethal doses of the parental strain Lm90SB2 and Lm90SB2-ΔinlK gene deletion strain were 10^5.74and 10^5.43,respectively,indicating that the inlK gene did not express significantly on the virulence of the Lm90SB2 strain;Comparing the bacterial load in mouse tissues,it was found that the bacterial load in the brain and spleen of the two increased with the prolongation of pathogenic time.It was higher than that of Lm90SB2 strain,but only the bacterial colonization number in spleen tissue at 72h showed extremely significant difference（P<0.01）,and there was no significant difference in brain tissue;After the neutralizer screening test,it was found that the PBS solution containing3g/L lecithin+3g/L Tween 80 and the PBS solution containing 5g/L sodium thiosulfate+5g/L lecithin+20g/L Tween 80 were composed of Neutralizers can neutralize the Neogermin and 84 disinfectants used in this test respectively;The sterilization rates of the two strains of bacteria suspensions were both 100%at 1,5,10 and 20 min by different proportions of Neogermin（1:15,1:30）and 84 disinfectant（1:50,1:100）;The results of the biofilm clearance test showed that the biofilm clearance rates of the Lm90SB2 strain were higher than those of the Lm90SB2-ΔinlK strain;The results of bactericidal rate test showed that the bactericidal rate of Lm90SB2-ΔinlK strain biofilm was higher than that of Lm90SB2 parental strain at 1,5and 10 min,and the bactericidal rate was 100%at 20 min.The results showed that the reduction of biofilm production and the incomplete spatial structure would lead to a significant increase in the bactericidal efficiency of the disinfectant against the Lm90SB2 strain.Conclusion:In this experiment,a stably inherited Lm90SB2-ΔinlK gene deletion strain was successfully constructed.By comparing the Lm90SB2 parent strain and the Lm90SB2-ΔinlK gene deletion strain biofilm formation ability,growth curve,hemolytic activity,median lethal dose LD50,brain,spleen tissue and organ load,it was found that the deletion strain and the parent strain have some biological functions.The differences indicated that the inlK gene had a certain regulatory effect on some biological functions of the Lm90SB2 strain.Especially the deletion of inlK gene can significantly reduce the biofilm formation ability of Lm90SB2 strain.The disinfectant sterilization rate test found that the Lm90SB2-ΔinlK gene deletion strain was more likely to be killed by disinfectants,which was directly related to the biofilm’s resistance to disinfectants.The bactericidal efficiency of the disinfectant was significantly enhanced due to the reduced biofilm formation ability of the Lm90SB2-ΔinlK gene deletion strain.Therefore,through the results of this study,it is expected to find a substance that can inhibit the expression of inlK gene to reduce the biofilm formation ability of Lm90SB2 strain,and lay a certain foundation and provide certain scientific reference for the effective prevention and control of LM pollution.